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Organophosphates (OPs) are important toxic compounds commonly used for a variety of purposes in agriculture, industry, and household settings (Maroni et al., 2000; Pope, 1999). These compounds affect a variety of central nervous system (CNS) functions (Pope et al., 2005). The relation between cognitive and behavioral effects following OPs intoxication has been well studied, and short-term neurobehavioral and emotional deficit in response to chlorpyrifos (CPF) exposure have been reported (Bushnell et al., 2001; Lopez-Crespo et al., 2009). Other studies have also reported neurobehavioral effects in humans after acute OP poisoning (Rohlman et al., 2011). In recent years, new evidence has pointed to the propensity of some OPs to cause neurotoxicity, neuroinflammation, and/or neurodegeneration (Ruiz-Munoz et al., 2011; Tansey et al., 2007), most likely via oxidative injury. It has also been demonstrated that acute administration of diisopropylphosphorofluoridate (DFP) to rats is associated with a significant increase in biomarkers of reactive oxygen species (ROS) (F2-isoprostanes [F2-IsoPs] and F4-neuroprostanes [F4-NeuroPs]) (Zaja-Milatovic et al., 2009). Other reports have demonstrated CPF-induced ROS generation (Gupta et al., 2010). In addition, acute CPF exposure in Wistar rats has been shown to cause short-term impairment in sensorimotor and cognitive functions, partly due to changes in brain lipoperoxidation (Ambali et al., 2010). Only a few studies have addressed the ability of acute parathion (PTN) exposure to elicit oxidative stress (Ojha and Srivastava, 2012). Given these observations and existing gaps in the literature, the major goal of this study was to investigate and compare the effects of CPF, DFP, and PTN on short-term cognitive and behavioral consequences, relating these effects to the respective efficacy of these OPs to generate oxidative stress. Acetylcholinesterase (AChE) inhibition and accumulation of acetylcholine at cholinergic synapses is evident within minutes to hours following exposure to OPs (Ecobichon, 1997; Lotti, 2001). However, the sensitivity of various AChE forms to OPs has yet to be determined. Within the brain, AChE exists in different molecular forms, mainly soluble (salt-soluble): monomeric, dimeric, and tetrameric forms, and particulate membrane-bound oligomeric (detergent-soluble). The tetrameric is the most abundant AChE form in the mammalian brain (Saez-Valero et al., 2003). Although the catalytic activity for both forms is analogous, it has been noted that they differ in mediating biological functions, such as plasticity (Kim et al., 2011) and cell adhesion properties (Ray et al., 2010); the latter are functions mediated by the AChE particulate form. Several studies have reported that in neurodegenerative disorders, such as Alzheimer’s disease (AD), the tetrameric form is selectively lost with a relatively small increase in AChE soluble monomers (Saez-Valero et al., 1999). Accordingly, the cholinergic system, and most specifically the AChE particulate form, plays an essential role in modulating cognitive performance in humans, vertebrates, and invertebrates. Therefore, emphasis should be placed on investigating the role of each of the AChE forms in relation to behavioral performance and other parameters. In addition, the recovery of AChE activity following OP exposure may result from increased expression of AChE causing variations in protein levels of each of the AChE forms providing additional rationale for studies aimed at deciphering their relative role in mediating various aspects of OP poisoning. Accordingly, we considered it timely to study the possible differences in OP-induced inhibition of these molecular forms. These observations led us to the second objective of this work, which is closely related to the first one, namely, is there a relationship between the inhibition of the soluble and particulate forms of AChE and oxidative stress? Furthermore, by inference, we questioned if CPF, DFP, and PTN mediate their effect by an analogous mechanism and similar time course? Therefore, a second experiment was designed to determine the degree of inhibition of AChE activity by separating the soluble and particulate forms of the enzyme. Several studies have suggested that protein product multimerization and membrane-bound properties of AChE (Meshorer and Soreq, 2006; Nijholt et al., 2004) as well as the recovery of AChE can be attributable to the alternative splicing of the AChE gene (Soreq and Seidman, 2001). In the CNS, Synaptic AChE-S mRNA is the main transcript expressed. Its product may be in the soluble form or it may be membrane-bound. However, under stress conditions, such as OPs exposure (Evron et al., 2007; Perrier et al., 2005), the transcription of the read-through splice variant, AChE-R mRNA increases (Adamec et al., 2008; Farchi et al., 2007). The AChE-R variant remains as a soluble monomer (Soreq and Seidman, 2001), and its overexpression may change the balance between the soluble and membrane-bound forms (Saez-Valero et al., 2003) and consequently their functions, affecting the restoration of cholinergic function. Finally, acylpeptide hydrolase (APH) has been considered a second target of OPs (Quistad et al., 2005; Rosenblum and Kozarich, 2003). The role of APH in the CNS is unknown. Notably, APH is significantly more sensitive to inhibition by diclorvos (DDVP) and DFP than AChE (Richards et al., 2000; Pancetti et al., 2007). APH participates in the degradation of oligomeric amyloid-beta, potentially representing a new therapy aimed at reducing neurodegeneration in the AD brain (Yamin et al., 2009). Conversely, inhibition of APH may induce neurodegeneration by increasing brain amyloid levels (Salazar et al., 2011). Accordingly, APH activity studies were incorporated into the study design to allow for comparison on the temporal dynamics of its inhibition by the three OPs with that of AChE.

Organophosphate compounds are cholinesterase inhibitors widely used in agriculture, industry, household products, and even as chemical weapons. Their major mechanism of acute toxic action is the inhibition of acetylcholinesterase, which is responsible for the degradation of the neurotransmitter acetylcholine. An organophosphorus ester-induced chronic neurotoxicity (OPICN) syndrome has been proposed. The OPICN syndrome could result from both long-term exposure to subclinical doses of OPs and after acute poisoning. Development of animal models for the cognitive decline are required and could later help to elucidate the mechanisms involved in this long-term effect on the central nervous system. Previously, we have found performance decrements in a four-trial repeated acquisition spatial task in a water maze. The present study includes two experiments to extend the long-term behavioral effects observed. Rats were injected either once or twice with chlorpyrifos (CPF) and then tested months after in a two-trial repeated acquisition task in a water maze. Our results confirm and extend the long-term behavioral effects of subcutaneous administration of CPF. The two treatments used produced performance decrements that suggest functional central nervous system alterations.

Organophosphate (OP) compounds are chemicals widely used in agriculture, industry and households and even as chemical weapons. The major mechanism of acute toxic action is the inhibition of acetylcholinesterase (AChE), which is responsible for the degradation of the neurotransmitter acetylcholine. A chronic OP-induced neuropsychiatric disorder (COPIND), which could result from both long-term exposure to subclinical doses of OP and after acute intoxication, has been proposed. These reports claim to develop animal models that could parallel behavioural and cognitive effects and that could later help to elucidate the mechanisms involved in this long-term affectation of the central nervous system. The present study uses a series of behavioural tests to discern the short- and long-term effects of acute intoxications with paraoxon (Px) or chlorpyrifos (CPF). Our results suggest that months after acute exposure to these OPs functional central nervous system alterations can be detected using a repeated acquisition spatial task in the water maze, for CPF, and in amphetamine-induced place preference paradigm, for both Px and CPF.

The majority of reports assessing opioid drug discrimination learning (DDL) have concentrated on characterizing the stimulus properties of compounds selective for mu and kappa opioid receptors. Assessments of delta opioid DDL have been limited and, to date, these assessments have been restricted to the monkey and pigeon. No assessment of delta stimulus control has been examined in rodents. To that end, the present experiment examined discriminative control by the selective delta agonist SNC80 in rats and its generalization to and antagonism by compounds relatively selective to the delta and mu receptor subtypes using the conditioned taste aversion baseline of DDL. Animals injected with 5.6 mg/kg of SNC80 prior to a saccharin-LiCl pairing and with the SNC80 vehicle prior to saccharin alone acquired the discrimination within seven conditioning cycles. The discriminative effects of SNC80 were maximal at 20 min, partial at 120 min, and lost at 240 min. The discrimination was dose dependent in that as the dose of SNC80 increased, the amount of saccharin consumed decreased. In subsequent generalization tests, the delta agonist SNC162 produced SNC80-appropriate responding at a dose of 18 mg/kg. Conversely, the mu agonist morphine produced vehicle-appropriate responding at all doses tested. These selective generalization patterns with SNC162 and morphine suggest that the discriminative effects of SNC80 are mediated at the delta, but not the mu, receptor, a conclusion supported by the fact that SNC80's discriminative control was completely blocked by the delta-selective antagonist NTI, but not by the mu-selective antagonist naltrexone. The present findings indicate that not only do rats readily discriminate both mu- and kappa-selective agonists from their respective vehicles, but they also discriminate compounds that are selective for the delta receptor subtype, thus extending the class of compounds that can serve such discriminative functions for the rat.

Organophosphates (OPs) affect behavior by inhibiting acetylcholinesterase (AChE). While the cognitive short-term effects may be directly attributed to this inhibition, the mechanisms that underlie OP's long-term cognitive effects remain controversial and poorly understood. Accordingly, two experiments were designed to assess the effects of OPs on cognition, and to ascertain whether both the short- and long-term effects of are AChE-dependent. A single subcutaneous dose of 250 mg/kg chlorpyrifos (CPF), 1.5 mg/kg diisopropylphosphorofluoridate (DFP) or 15 mg/kg parathion (PTN) was administered to male Wistar rats. Spatial learning was evaluated 72 h or 23 weeks after exposure, and impulsive choice was tested at 10 and 30 weeks following OPs administration (experiment 1 and 2, respectively). Brain soluble and membrane-bound AChE activity, synaptic AChE-S mRNA, read-through AChE-R mRNA and brain acylpeptide hydrolase (APH) activity (as alternative non-cholinergic target) were analyzed upon completion of the behavioral testing (17 and 37 weeks after OPs exposure). Both short- and long-term CPF treatment caused statistically significant effects on spatial learning, while PTN treatment led only to statistically significant short-term effects. Neither CPF, DFP nor PTN affected the long-term impulsivity response. Long-term exposure to CPF and DFP significantly decreased AChE-S and AChE-R mRNA, while in the PTN treated group only AChE-S mRNA levels were decreased. However, after long-term OP exposure, soluble and membrane-bound AChE activity was indistinguishable from controls. Finally, no changes were noted in brain APH activity in response to OP treatment. Taken together, this study demonstrates long-term effects of OPs on AChE-S and AChE-R mRNA in the absence of changes in AChE soluble and membrane-bound activity. Thus, changes in AChE mRNA expression imply non-catalytic properties of the AChE enzyme.

Chlorpyrifos (CPF) is an organophosphate (OP) insecticide that is metabolically activated to the highly toxic chlorpyrifos oxon. Dietary exposure is the main route of intoxication for non-occupational exposures. However, only limited behavioral effects of chronic dietary exposure have been investigated. Therefore, male Wistar rats were fed a dose of 5mg/kg/day of CPF for thirty-one weeks. Animals were evaluated in spatial learning and impulsivity tasks after 21 weeks of CPF dietary exposure and one week after exposure ended, respectively. In addition, the degree of inhibition of brain acetylcholinesterase (AChE) was evaluated for both the soluble and particulate forms of the enzyme, as well as AChE gene expression. Also, brain acylpeptide hydrolase (APH) was investigated as an alternative target for OP-mediated effects. All variables were evaluated at various time points in response to CPF diet and after exposure ended. Results from behavioral procedures suggest cognitive and emotional disorders. Moreover, low levels of activity representing membrane-bound oligomeric forms (tetramers) were also observed. In addition, increased brain AChE-R mRNA levels were detected after four weeks of CPF dietary exposure. However, no changes in levels of brain APH were observed among groups. In conclusion, our data point to a relationship between cognitive impairments and changes in AChE forms, specifically to a high inhibition of the particulate form and a modification of alternative splicing of mRNA during CPF dietary exposure.

This study examined the time-course effects (2, 7, 14 and 30 days) of acute chlorpyrifos (CPF) intoxication (250 mg/kg, s.c.) on monoamine systems and acetylcholinesterase (AChE) activity in the striatum and nucleus accumbens of adult male rats. We show that CPF produced significant long-term inhibition of AChE activity in the striatum and nucleus accumbens. In the striatum, CPF intoxication resulted in changes in dopamine (DA) metabolism after 2 days and changes in serotonin (5-HT) turnover after 7 and 15 days. Significant decreases in monoamine content including norepinephrine (NE), DA, 5-HT and their metabolites were found in the nucleus accumbens 30 days after CPF intoxication. These results suggest that acute exposure to CPF induces long-term changes in the monoamine systems (NE, DA and 5-HT) in adult animals. The lack of correlation between regional AChE activity and neurochemical outcomes points to independent mechanisms., This study was supported by research projects SEJ2006-15628-C02-01/PSIC, SEJ2006-15226-C02-01/PSIC and FIS061212.

Based on previous data from elevated plus-maze tests suggesting a possible anxiogenic effect of the insecticide chlorpyrifos (CPF), the experiment reported here was designed to determine whether this organophosphate (OP) caused an interoceptive discriminative stimulus (IDS) in rats similar to that produced by the anxiogenic drug pentylenetetrazol (PTZ). Rats were trained to discriminate PTZ (20 mg/kg) from saline, using a drug discrimination procedure. When appropriate lever selection was achieved, generalization tests were performed. Tests of various doses of PTZ showed that the drug exerts dose-dependent discriminative control over response. Two more generalization tests were conducted with 250 mg/kg of CPF and 76.8 mg/kg of LiCl for up to 9 days. Results revealed that CPF (250 mg/kg s.c.) produced a PTZ-like IDS that fully substituted for PTZ 24 h after injection and that subjective effects remain for at least 6 days. However, administration of LiCl did not produce any generalization to PTZ on any of the days tested. These results suggest that CPF shares a site of action, and perhaps functional properties, with PTZ that last for several days, are not due to general malaise and should be taken into account in the use of cholinesterase inhibitors in the treatment of different types of dementia.

Organophosphates (OPs) are important toxic compounds commonly used for a variety of purposes in agriculture, industry and household settings. It has been well established that the main mechanism of acute toxic action of OP is the inhibition of acetylcholinesterase (AChE). However, we observed long term deficit after acute subcutaneous exposure to Chlorpyrifos (CPF) even when AChE activity is restored. In fact, besides AChE inhibition, non-AChE targets have also been proposed as an alternative mechanism involved in the acute lethal action and side effects of short or long-term exposure. In this context, our main aim in this research was to establish a dose-response curve of Acylpeptide hydrolase (APH) and AChE regional brain activity after acute CPF administration that could explain these long term effects observed in the literature. Moreover, since available data suggest that long term effects of OPs exposure could involve neuronal cell death, our second aim was to evaluate, assessing by Fluoro-Jade B (FJB) staining, whether CPF produces induced cell death. Our results show that an acute exposure to 250 mg/kg CPF does not induce neuronal death as measured by FJB but produces highest AChE regional brain inhibition after administration. In addition, APH seems to be more sensitive than AChE to CPF exposure because after 31 days of exposure, complete recovery was seen only for APH activity at Frontal Cortex, Cerebellum and Brain Stem.